PROJECT SUMMARY/ABSTRACT Urinary leakage of protein (proteinuria) is a prominent feature of numerous kidney diseases that independently predicts mortality and progression to end-stage kidney disease. Proteinuria stimulates sodium retention in the kidney?s tubule, contributing to extracellular fluid volume retention, edema, and increased blood pressures. Inhibition of the epithelial sodium channel (ENaC) in the distal renal tubule reduces sodium retention associated with proteinuria. Thus, ENaC likely contributes to proteinuria-induced sodium retention. Experimentally applied proteases cleave and activate ENaC. Among the proteases capable of activating ENaC is the serum protease, plasmin. In individuals with proteinuric kidney disease or severe hypertension, damaged glomeruli leak serum plasmin or its precursor, plasminogen into the urine. This mentored career development award explores the hypothesis that, in proteinuric kidney disease, serum proteases such as plasmin leaked into the kidney tubule activate ENaC, promoting sodium reabsorption, fluid retention, and increased blood pressure. This proposal will explore this hypothesis using both clinical data and animal modeling. The first aim examines whether urinary plasmin/plasminogen is associated with increased blood pressure in people with type 1 diabetes. Individuals with type 1 diabetes are susceptible to proteinuria secondary to diabetic nephropathy. Dr. Ray will examine previously collected urine specimens from a longitudinal study of subjects with type 1 diabetes. He will determine whether urine plasmin/plasminogen correlates independently with increases in blood pressure or hypertension and whether urinary plasmin/plasminogen precedes hypertension. The second aim examines whether ENaC is a key target of proteolytic cleavage contributing to sodium retention in proteinuric kidney disease. Dr. Ray will establish a mouse line expressing genetically modified ENaC. This modification renders ENaC resistant to full activation by proteases, including plasmin. Dr. Ray will determine whether these mice experience reduced proteinuria-induced sodium reabsorption and fluid retention, and lower blood pressures than mice expressing wild-type ENaC. This study will define the role of ENaC in proteinuria- associated sodium retention and determine whether blockers of ENaC or proteases that activate ENaC may be of future clinical utility in treating individuals with proteinuric kidney disease. Dr. Ray will use these aims as a mechanism to accomplish his short-term goal of achieving scientific independence and launching a research group and his long-term goal of growing into a leading physician scientist in electrolyte handling and hypertension. This proposal?s animal-based aim will provide Dr. Ray with experience in the use of animal models to study electrolyte physiology including (i) hands-on experience in building transgenic mouse strains, (ii) experience with animal breeding and colony management, (iii) protein biochemistry, (iv) tissue microscopy, (v) measurement of electrolyte balance and blood pressures in mice, and (vi) measurement of electrolyte flux and ion channel activity in isolated kidney tubules. Workshops in mouse colony maintenance at the Jackson Laboratories and mouse phenotyping at Johns Hopkins will reinforce these skills. This proposal?s translational aim will provide Dr. Ray with the skills to explore the implications of basic sciences research in human subjects. He will gain experience analyzing clinical data and use of human specimens. He will learn how to use biostatistical tools to account for confounders and missing data in clinical research. Coursework in regression analysis and biomarker studies will augment these skills, which will provide a foundation to translate his physiologic insights into improved human health. For the duration of this award, Dr. Ray will maintain his familiarity with clinical medicine by devoting up to 25% of his efforts to teaching and clinical care of patients and by attending yearly medical conferences. The University of Pittsburgh and the Pittsburgh Center for Kidney Research provide Dr. Ray with an environment rich in resources and intellectual stimulation for his training and for the completion of this project. A superb group of mentors will oversee this career development award. Tom Kleyman, MD, this award?s primary mentor, is an international leader in renal electrolyte physiology, director of the Pittsburgh Center for Kidney Research and mentor to eight career development awardees and five R01 recipients within the last decade. Co-mentor Mark Unruh, MD, heads the University of New Mexico?s Division of Nephrology and has extensive experience is clinical trials and outcomes research. Co-mentor Becky Hughey, PhD has extensive experience examining proteolytic cleavage of ENaC and has mentored scientific trainees for 40 years. Co- mentor Ed Jackson, PhD directs the Pittsburgh Center for Kidney Research Metabolomics Core, with extensive experience in examination of rodent hemodynamics and over 50 past trainees. This group of mentors will ensure the success of this proposal and the development of Dr. Ray into an independent physician scientist with the skills to further our understanding of the physiology of electrolyte handling and the ability to translate those insights into clinical advances.